Biophysical analysis of the Mycobacteria tuberculosis peptide binding protein DppA reveals a stringent peptide binding pocket

Abstract

The peptide binding protein DppA is an ABC transporter found in prokaryotes that has the potential to be used as drug delivery tool for hybrid antibiotic compounds. Understanding the motifs and structures that bind to DppA is critical to the development of these bivalent compounds. This study focused on the biophysical analysis of the MtDppA from M. tuberculosis. Analysis of the crystal structure revealed a SVA tripeptide was co-crystallized with the protein. Further peptide analysis demonstrated MtDppA shows very little affinity for dipeptides but rather preferentially binds to peptides that are 3-4 amino acids in length. The structure-activity relationships (SAR) between MtDppA and tripeptides with varied amino acid substitutions were evaluated using thermal shift, SPR, and molecular dynamics simulations. Efforts to identify novel ligands for use as alternative scaffolds through the thermal shift screening of 35,000 compounds against MtDppA were unsuccessful, indicating that the MtDppA binding pocket is highly specialized for uptake of peptides. Future development of compounds that seek to utilize MtDppA as a drug delivery mechanism, will likely require a tri- or tetrapeptide component with a hydrophobic -non-acidic peptide sequence.

Keywords: ABC; DppA; Peptide transport; SPR; Thermal denaturation; Tuberculosis.

Figure 1.

MtDppA with SVA bound. The structure of MtDppA with the SVA peptide as aqua sticks. Two dimensional ligand interaction diagram generated in MOE.

Figure 2.. The binding characteristics of SVAA.

(A) The linear regression plot of 2-fold dose response for SVA and SVAA (B) SPR sensorgrams show nanomolar affinities of SVAA and SVA.

Figure 3.. MtDppA is an oligopeptide binder.

(A) MtDppA showed no binding to di-peptides while tri and tetra peptides stabilized MtDppA ≥1°C across the dose response range. Peptides were tested in triplicate and error bars represent SD. (B) Millimolar amounts of AA are required to stabilize MtDppA

Figure 4. Surface plasmon resonance sensorgrams profiling peptide length.

Peptide binding to MtDppA indicate dipeptides do not bind to MtDppA while tri- and tetrapeptides do.

Figure 5.

Substitution of aspartic acid modulates peptide binding to MtDppA. (A) Thermal denaturation study of aspartic acid substitution (B) SPR Sensorgrams of aspartic acid substitutions

Figure 6.

Substitution of lysine modulates peptide binding to MtDppA. (A) Thermal denaturation study of lysine substitutions (B) SPR Sensorgrams of lysine substitutions

Figure 7:. Analysis of 50ns MD simulation of MtDppA bound to tripeptides.

A) The room mean square deviation (RMSD) of the MtDppA backbone during the 50ns MD simulation for SVA (black), VKV (blue), VDV (orange), and VVV (green). B) The calculated free energy of binding (ΔG) using the MM/GBSA method.

Figure 8 -

Rifapentine vs Rifampin binding to MtDppA. (A) Structures of rifapentine and rifampin (B) Stabilization of MtDppA by rifapentine >2°C, rifampin does not interact with MtDppA. (C) SPR sensorgrams from rifapentine (left) and rifampin (right) showing significant differences in binding activity.